The long-range objective is to clarify the origin of "resident" alveolar macrophages and the nature of factors regulating production of these unique lung cells that form the first line of defense against inhaled particles and microorganisms. It is known that in organ culture, embryonic mammalian lungs generate a replicating population of phagocytes indistinguishable from alveolar macrophages by morphological, functional, and immunocyto- chemical criteria. These cells are termed "premedullary" because they appear prior to formation of the bone marrow and in the absence of circulating monocytes. Specific aims of the proposed program are 1) to define the cell type or types that give rise to premedullary macrophages; 2) to clarify events occurring during transition from precursor to macrophage; 3) to determine the population kinetics and replicative capacity of alveolar macrophages having a premedullary origin; and 4) to identify local sources of factors which may trigger transformation of precursors and/or act to maintain the resulting differentiated phagocytes. Relative to aims 1 and 2, embryonic rat and hamster lungs will be fixed for transmission electron microscopy at short intervals during the first 24 hr. in organ culture. Thin sections will be surveyed for number and types of potential leukocyte precursors and for transitional forms between precursor and mature phagocyte. Nascent macrophages will be traced back as closely as possible to their origin b" enzyme cytochemistry (acid phosphatase, NADPH oxidase) and immunocytochemical localizations of leukocyte and macrophage-specific marker proteins. Relative to aim 3, organ cultured lungs will be grown on medium containing 3H-thymidine and processed for light microscopic autoradiography. Cell cycle times and replication rates will be determined after continuous labeling by use of standard statistical methods. Respecting aim 4, organ cultured lungs and lung explants will be examined immunocytochemically to localize factors known to influence macrophage differentiation and proliferation in other systems. These include hematopoietic colony-stimulating factors (CSFs), platelet-derived growth factor, and hemopoietin-l. Efforts will also be made to influence macrophage production and growth within explanted lungs and on adjacent culture medium by direct application of CSFs. Results of these studies will be evaluated by transmission electron microscopy and by 3H-thymidine labeling and light microscopic autoradiography of Giemsa-stained 2 mu m glycol methacrylate sections.